Investigation of Babesia spp. and Theileria spp. in ticks from Western China and identification of a novel genotype of Babesia caballi

Babesia spp. and Theileria spp. are tick-borne protozoan parasites with veterinary importance. In China, epidemiological and genetic investigations on many Babesia and Theileria species were still absent in many areas and many tick species. From Aug 2021 to May 2023, 645 ticks were collected from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers in seven counties in three provinces including Xinjiang (Qitai, Mulei, Hutubi, and Shihezi counties), Chongqing (Youyang and Yunyang counties), and Qinghai (Huangzhong county). Three tick species were morphologically and molecularly identified (334 Hyalomma asiaticum from Xinjiang, 245 Rhipicephalus microplus from Chongqing, and 66 Haemaphysalis qinghaiensis from Qinghai). A total of three Babesia species and two Theileria species were detected targeting the 18S gene. The COI and cytb sequences were also recovered from Babesia strains for further identification. In R. microplus from Chongqing, Babesia bigemina, the agent of bovine babesiosis, was detected. Notably, in H. asiaticum ticks from Xinjiang, a putative novel genotype of Babesia caballi was identified (0.90%, 3/334), whose COI and cytb genes have as low as 85.82% and 90.64–90.91% nucleotide identities to currently available sequences. It is noteworthy whether the sequence differences of its cytb contribute to the drug resistance of this variant due to the involvement of cytb in the drug resistance of Babesia. In addition, Theileria orientalis and Theileria annulata were detected in R. microplus from Chongqing (12.20%, 31/245) and H. asiaticum from Xinjiang (1.50%, 5/334), respectively. These results suggest that these protozoan parasites may be circulating in domestic animals in these areas. The pathogenicity of the novel genotype of B. caballi also warrants further investigation. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-024-04171-z.


Introduction
Babesia and Theileria spp.belonging to the phylum Apicomplexa are protozoan parasites with veterinary importance.They are distributed worldwide and cause significant economic losses in husbandry, especially in the tropic and subtropic areas [1].To date, more than 100 species of Babesia and Theileria have been recorded worldwide, many of which are pathogens for humans and domestic animals.For example, Babesia ovis, B. motasi, B. crassa, B. foliata, B. taylori are common agents of ovine babesiosis [2], while B. caballi, Theileria equi, and T. haneyi usually infect equines [3].They are also frequently detected in wild animals.For example, Babesia spp.have been detected in Alpine chamois, roe deer, red deer, and wild boar, while Theileria spp.were identified in foxes, wild boar, and red deer [4].These parasites parasitize in erythrocytes and lymphocytes of their animal hosts, resulting in loss of body weight, milk output, and even animal death [5].Some of them are also considered agents of emerging zoonosis, such as B. divergens and B. microti [6].
Ticks, a group of obligatory hematophagous arthropods usually infest domestic and wild animals, are the major vectors of Babesia and Theileria spp.To date, more than 21 tick species, mainly hard ticks, have been reported to transmit these parasites [7].Meanwhile, asymptomatic animals usually act as their reservoirs.In China, at least Theileria species (T.ovis, T. orientalis, T. sinensis, T. uilenbergi, T. luwenshuni, and T. annulata) have been detected in various ticks and animals [8].However, compared to many other tick-borne pathogens (Rickettsia, Anaplasma, Coxiella, Borrelia, etc.), these tick-borne protozoan parasites are still largely neglected and unstudied in many areas and many tick species in China.Genetic investigations were also absent for many Babesia and Theileria species.In this study, we collected three tick species from three provinces in Western China, and investigated the genetic diversity of Babesia spp.and Theileria spp. in them.

Sample collection, DNA extraction, and identification of the ticks
From Aug 2021 to May 2023, a total of 645 ticks were collected from the body surface of domestic animals in seven locations (counties, districts, or county-level cities) in three provinces: Qitai, Mulei, Hutubi, and Shihezi counties in Xinjiang Uygur Autonomous Region (from cattle, goats, and camels); Yunyang and Youyang counties in Chongqing Municipality (from cattle); Huangzhong county in Qinghai Province (from goats) (Fig. S1).Ticks were carefully removed from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers.Probability sampling was used in this study.First of all, the collected ticks were morphologically identified into species according to their morphological features including the capitula, legs, anal groove, etc. [9,10].After washing twice with PBS, the ticks were subjected to DNA extraction using the TIANamp genomic DNA Extraction Kit (TIANGEN company), and then the COI gene sequences were amplified for molecular confirmation of the tick species (Primers shown in reference [11]).
Detection and identification of the Babesia spp.and Theileria spp.
All the 645 DNA samples were screened for Babesia spp.and Theileria spp.using primers amplifying a conserved region of the 18S gene, yielding an approximately 600-650 bp product.Primer pairs of New-Babesia-F/ New-Babesia-R1 and New-Babesia-F/New-Babesia-R2 were designed in this study (our lab designed all the primers and the sequences are shown in Table S1).The PCR conditions of first round PCR reaction: 94 °C for 3 min, 40 cycles of denaturation at 94 °C for 40 s, annealing at 50 °C for 40 s, and extension at 72 °C for 60 s, with a final extension at 72 °C for 8 min.The PCR conditions of second round PCR reaction: 94 °C for 3 min, 40 cycles of denaturation at 94 °C for 40 s, annealing at 48 °C for 40 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 8 min.The PCR products were then subjected to sequencing in Sangon Company.The obtained sequences were then aligned with reference sequences in the GenBank Database by BLASTN (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi) to determine their genus and species initially.
For further identification of the detected Babesia strains, the mitochondrial cytb (cytochrome b) and COI (cytochrome oxidase subunit I) genes were PCR amplified from randomly selected samples which are positive for each Babesia species.The primers used were shown in the reference [12].The PCR conditions of cytb: 94 °C for 5 min, 40 cycles of denaturation at 94 °C for 40 s, annealing at 55 °C for 40 s, and extension at 72 °C for 90 s, with a final extension at 72 °C for 8 min.The PCR conditions of COI: 94 °C for 5 min, 40 cycles of denaturation at 94 °C for 40 s, annealing at 60 °C for 40 s, and extension at 72 °C for 70 s, with a final extension at 72 °C for 8 min.

Genetic and phylogenetic analysis of the sequences
All the recovered nucleotide sequences were compared with references in the GenBank Database analyzed by BLASTN (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).Most, if not all, formally validated Babesia and Theileria species were selected as reference sequences.Along with reference sequences, the sequences were manually aligned by the ClustalW method in the MEGA program.Maximum Likelihood (ML) trees were reconstructed in the GTR model using PhyML v3.0 [13].The substitution model test was performed to select the best-fit phylogenetic model.The confidence values for each branch of the trees were determined by bootstrap analysis with 100 repetitions, and the confidence values larger than 70 were considered adequate.All the phylogenetic trees were mid-point rooted.

Determination of the tick species
All the 334 ticks from Xinjiang were identified to be Hyalomma asiaticum.Meanwhile, the 245 ticks from Chongqing and 66 ticks from Qinghai were identified to be Rhipicephalus microplus and Haemaphysalis qinghaiensis, respectively.The detailed information of ticks is shown in Table 1.All the tick species were molecularly confirmed after morphological identification.The COI sequences were all > 99% identical to those in the Gen-Bank Database.
Notably, a B. caballi variant was identified in H. asiaticum ticks from Qitai (1.67%, 2/120) and Mulei (1.39%, 1/72) counties in Xinjiang.Although its 18S gene showed 100% identity to some previously reported B. caballi strains, the COI and cytb genes have as low as 85.82% and 90.64-90.91%nucleotide identities to the B. caballi strain from Japan (Accession numbers for the COI and cytb genes: AB499086.1).In the phylogenetic trees, they also form relatively independent clades (Fig. 1).Therefore, we suppose that they represent a previously uncharacterized novel genotype of B. caballi.
No H. qinghaiensis ticks from Qinghai Province were positive for Babesia.All the obtained sequences have been uploaded to the GenBank Database (Table S2).

Discussion
Among the three detected Babesia species, B. bigemina is a worldwide distributed agent of bovine babesiosis.It was also reported to infect other animals such as goats [14].In China, B. bigemina has been reported in ticks (mainly R. microplus) and domestic animals from many provinces including Gansu, Yunnan, Guangxi, Chongqing, Shandong, Liaoning, Henan, Hubei, and Xinjiang, suggesting its wide geographic distribution [15].Actually, it is considered one of the most prevalent agents causing bovine babesiosis in China [16].In this study, B. bigemina was detected in R. microplus ticks collected from cattle in Chongqing.Although the positive rate is low, it suggests that B. bigemina may be circulating in cattle in this area.
An unnamed Babesia species was identified in H. asiaticum from Xinjiang, with all its sequences (18S, COI, and cytb) highly homologous to Babesia strains previously identified in sheep from Xinjiang (DQ159073.1,MK962313.1,MK962313.1).Interestingly, all H. asiaticum ticks positive for this Babesia were removed from cattle, camels, and goats.This suggests two possibilities: First, the Babesia sp. may be from the blood meal of infected cattle, camels, and goats.Namely, it may be pathogenic to these domestic animals.Second, H. asiaticum ticks might be the vector of this Babesia and play a role in its transmission or maintenance.In future studies, the pathogenicity of this Babesia to domestic animals still warrants further investigation.
It is out of our expectation that a novel genotype of B. caballi was identified in H. asiaticum ticks from Xinjiang.Babesia caballi is the agent of equine piroplasmosis, an economically important tick-borne disease worldwide [17].The infection of B. caballi in horses usually causes anemia, hemoglobinuria, fever, abdominal inflammation, weakening, etc. [18].In China, some molecular and serological investigations targeting B. caballi have been carried out, reporting its presence in multiple provinces including Xinjiang, Jilin, and Gansu [19][20][21].Although reports on B. caballi is common, further analysis such as genotyping has been rare.Most studies are preliminary and very few references were available except for the 18S gene.In this study, we identified a novel genotype of B. caballi, whose cytb and COI sequences show low homologies to existing sequences of B. caballi.The phylogenetic trees also show remarkable distances with other B. caballi strains and currently known Babesia species.The amino acid sequences of cytb and COI genes were also analyzed.The cytb sequences have highest 97.80% identity to B.caballi from Japan (GIX66475.1),while the COI sequences show 91.39% identity to reference B. caballi strains (BAI66165.1).This result confirmed that it represents a novel genotype.We suppose that the sequence difference may be due to the absence of enough reference sequences in the GenBank Database.As more sequences of B. caballi will be obtained in the future, we believe more genotypes may be identified.Besides, as was previously reported, the cytb gene is involved in the drug resistance of Babesia [22][23][24].It is noteworthy that the sequence differences contribute to the drug resistance of this variant.
Two Theileria species were detected in this study.Of those, T. annulata is the most pathogenic species in cattle causing tropical theileriosis [25], while T. orientalis is the most prevalent Theileria species in China [26].Both these two species are of economic importance in husbandry and veterinary.Ticks were considered vectors of Theileria.In this study, T. annulata was only detected in H. asiaticum, and T. orientalis was only identified in R. microplus ticks.However, the vector roles of both H. asiaticum and R. microplus are still to be determined, despite the frequent reports of Theileria in them [27][28][29][30][31].For T. orientalis, although its DNA has been detected in eggs of R. microplus tick [32], suggesting the existence of transovarial transmission, a study performed by Ghafar et al. [33] indicated that R. microplus fails to transmit T. orientalis.Notably, in this study, all the Theileria-positive ticks were fully engorged and were collected from cattle, indicating that the Theileria DNA might be from cattle blood.Namely, T. annulata and T. orientalis may be circulating in cattle in these areas.Furthermore, the phylogenetic tree also shows that the detected T. orientalis strains were divided into two clades (Fig. 2), suggesting its genetic diversity in this area.The characteristics of other genes, and their relational virulence and drug resistance still warrant further investigations.

Conclusion
A total of three Babesia species (B.bigemina, B. caballi, and Babesia sp.) and two Theileria species (T.orientalis and T. annulata) were detected in 645 ticks representing three species (H.asiaticum, R. microplus, and H. qinghaiensis) from western China.Of those, a novel genotype of B. caballi was identified.These proved the wide circulation and remarkable genetic diversity of Babesia and Theileria in China.

Table 1
Prevalence of Babesia spp.and Theileria spp. in ticks from three provinces of China